传染性法氏囊病病毒多表位模拟肽串联基因的表达与免疫原性分析

Expression and immunogenic analysis for the tandem-arranged multiple mimic epitope gene of Infectious Bursal Disease Virus

摘要:

用5株传染性法氏囊病病毒（IBDV）单克隆抗体（mAb）从噬菌体随机肽库中得到了5个含有不同IBDV抗原表位的模拟肽序列。在此基础上，将5个抗原表位用GGGS四肽连接构建多表位基因5epis。将该基因合成、克隆后，构建原核表达质粒pET-5epis，在大肠杆菌中表达重组多表位蛋白r5EPIS，经SDS-PAGE分析，r5EPIS占菌体总蛋白的15%、分子量10kDa。用IBDV单克隆抗体和多克隆抗体对r5EPIS进行免疫印迹对比分析，结果表明，r5EPIS具有IBDV特异性和免疫反应性。将r5EPIS经皮下注射免疫兔，400μg/只/次，免疫2次，间隔7d，用IBDV间接ELISA检测血清抗体，第一次免疫7d后抗体效价为1∶4000，第二次免疫14d后抗体效价升高到1∶256000，说明r5EPIS可诱导机体产生IBDV特异性抗体。用r5EPIS加免疫佐剂经肌肉注射免疫鸡，50μg/只/次，免疫2次后，血清抗体效价可达到1∶12800，用200个ELD50IBDV超强毒株GX8/99攻击实验鸡，r5EPIS免疫组全部存活，而单用佐剂对照组的死亡率为86.7%（13/15），证明r5EPIS可诱导机体产生抗IBDV感染的保护性免疫应答，预示构建的5epis可作为IBD多表位疫苗研究的候选基因。

Abstract:

Five mimic epitopes of Infectious bursal disease virus (IBDV) have been identified from a 12-mer phage-displayed peptide library by 5 monoclonal antibodies. Based on the sequences of the five epitopes, multiple epitope gene 5epis was constructed by the five epitopes being tandemly arranged and linked with 4-peptide GGGS. The expression plasmid pET-5epis was constructed and successfully expressed in E. coli. The resultant protein of 5epis was called r5EPIS.The results from SDS-PAGE analysis showed that the proportion of r5EPIS was 15% of the total bacterial proteins and the molecular weight of r5EPIS was 10kDa. By use of parallel immunoblotting test with corresponding monoclonal and polyclonal antibodies, the immunological specificity and reactivity of r5EPIS against IBDV have been verified. Rabbits were subcutaneously injected with r5EPIS (400μg per injection), twice with 7 days interval. The titers of the IBDV-specific antibody measured by indirect ELISA were up to 1∶4000 at the 7th day after first immunization and 1∶256000 at the 14th day after the second immunization. To determine the protective ability of r5EPIS to the challenge of IBDV, chickens were injected intramuscularly with r5EPIS in adjuvant twice with 7 days interval (50μg per injection) and the resultant antibody titer was up to 1∶12800 at the 7^th day after the second immunonization. After challenge with 200ELD₅₀ of virulent IBDV GX8/99 strain, all the chicken in r5EPIS-immunized group were survived in contrast to the mortality of 86.7%(13/15) in adjuvant control group, suggesting that r5EPIS had a potent ability to generate protective immune response and it implied that the constructed gene 5epis is a prospective candidate for the development of epitope-based IBD vaccine.